Protein synthesis with a cell-free protein synthesis system has been developed not only in a basic study but also in a practical field such as molecular diagnostics and high-throughput discovery of drug target. In recent years, several techniques of drastically increasing the amount of protein synthesis in this system have been explored (Japanese Patent Kokoku Publication No. JP-B-7-110236, Japanese Patent Kokai Publication No. JP-A-4-200390). Accordingly, this system has been utilized in mass production of proteins for structural analysis by X-ray crystallography, Nuclear Magnetic Resonance (NMR) or the like.
As an extract for carrying out the translation reaction, several extracts derived from E. coli, wheat germ, and rabbit reticulocyte are commercially available. In an E. coli extract, it is known that transcription-translation coupled reaction can be used for synthesizing a protein directly from a DNA. For example, a method using an E. coli S30 extract has been systematically developed by Zubay et al. (Geoffrey Zubay, Annual Review of Genetics, 1973, vol. 7, p. 267-287). The S30 extract comprises ribosomes necessary for translation of mRNA, aminoacyl tRNA synthetases, initiation factors (IF), elongation factors (EF) and release factors (RF) of peptide chain synthesis. When a DNA template is used for protein synthesis, a DNA construct, in which a target protein gene is inserted downstream of a strong promoter (generally, a T7 promoter), is added in the system together with a T7 RNA polymerase and four types of ribonucleotides (ATP, GTP, CTP and UTP) to couple both reactions of transcription and translation. Due to the requirement for ATP energy for synthesis of an aminoacyl-tRNA and a translation reaction with mRNA, an energy regeneration system such as a creatine kinase-creatine phosphate system is added to the cell-free system. With the above components, a protein synthesis reaction occurring in cells is reconstructed in vitro.
In the cell-free protein synthesis system using the S30 extract, various factors that influence the protein synthesis yield are known. For example, proteases present in the extract degrade synthesized proteins. In order to minimize the proteolytic degradation, various E. coli strains (for example, E. coli strain B) deficient in OmpT and Lon proteases have been produced.
On the other hand, various nucleases present in the extract degrade a template DNA or its transcription product, mRNA. With respect to the template DNA, the methods of cell-free protein synthesis are roughly classified into two types, one using a circular DNA cloned in a plasmid, λ phage or the like and another using a linear DNA of a PCR product or the like. Generally, a circular DNA is less susceptible to degradation with nucleases, and the protein synthesis yield is high. Nevertheless, in studies of post genomic research in recent years, structures and functions of a large number of proteins have been comprehensively analyzed, and improvement in production of proteins and efficient operation have been essential subjects. Accordingly, a system has been highly demanded in which a large number of linear DNAs are synthesized by PCR amplification with genomic DNA as a template and proteins are synthesized efficiently with a cell-free protein synthesis system using these DNAs.
It has been reported that the linear template DNA is susceptible to degradation with an endogenous exonuclease present in an E. coli extract (Pratt et al., Nucleic Acids Res., 9, 4459-4474, (1981); Benzinger et al., J. Virol., 15, 861-871, (1975); Lorenz and Wackernagel, Microbiol Rev., 58, 563-602, (1994)). It has also been known that since a protein complex called a degradosome recognizes and degrades an RNA, the expression efficiency is decreased. For solving these problems, a freeze-thawing procedure has been conducted as a step of producing an S30 extract to remove the degradosome (WO Pamphlet No. 01/83805), or an E. coli strain in which a mutation is introduced into the rne gene encoding an endonuclease RNase E as an essential component of the degradosome has been produced (for example, a strain BL21-Star, Invitrogen). Regarding the enzyme that degrades a linear DNA, DNA exonucleases such as RecBCD are considered to be a cause of degradation, and various mutant E. coli strains of RecBCD deficient in some of these nucleases have been produced (Yang et al., PNAS 77, 7029-7033 (1980)). However, despite that the degradation activity of linear DNAs in E. coli mutant strains of RecBCD has been decreased, the growth capability of the mutant strains is also lower in many cases. Thus, the strains are not necessarily appropriate for production of an extract for cell-free protein synthesis (Yu et al., PNAS, 97, 5978-5983, (2000)).